Temporal blastemal cell gene expression analysis in the kidney reveals new Wnt and related signaling pathway genes to be essential for Wilms' tumor onset

Abstract

Wilms' tumors (WTs) originate from metanephric blastema cells that are unable to complete differentiation, resulting in triphasic tumors composed of epithelial, stromal and blastemal cells, with the latter harboring molecular characteristics similar to those of the earliest kidney development stages. Precise regulation of Wnt and related signaling pathways has been shown to be crucial for correct kidney differentiation. In this study, the gene expression profile of Wnt and related pathways was assessed in laser-microdissected blastemal cells in WTs and differentiated kidneys, in human and in four temporal kidney differentiation stages (i.e. E15.5, E17.5, P1.5 and P7.5) in mice, using an orthologous cDNA microarray platform. A signaling pathway-based gene signature was shared between cells of WT and of earliest kidney differentiation stages, revealing genes involved in the interruption of blastemal cell differentiation in WT. Reverse transcription-quantitative PCR showed high robustness of the microarray data demonstrating 75 and 56% agreement in the initial and independent sample sets, respectively. The protein expression of CRABP2, IGF2, GRK7, TESK1, HDGF, WNT5B, FZD2 and TIMP3 was characterized in WTs and in a panel of human fetal kidneys displaying remarkable aspects of differentiation, which was recapitulated in the tumor. Taken together, this study reveals new genes candidate for triggering WT onset and for therapeutic treatment targets.

Figure 1

Wilms tumor signature set. (a) Expression behavior of the 18 genes during nephrogenesis in mouse. ↓ and ↑ indicate downregulation and upregulation in Wilms tumor, respectively, in comparison with differentiated kidney at transcriptional level. (b) Unsupervised hierarchical clustering based on expression pattern of the 18 genes. Black lines correspond to 90–100% of reliability assessed by Bootstrap. (c) Comparison of cDNA microarray and RT-qPCR experiments. (^*) Represents differentially expressed genes assessed by RT-qPCR that fulfilled cutoff criteria (fold change >3 and P<0.05). (d) Legend of colors. FK, fetal kidney; WT, Wilms tumor; DK, differentiated kidney; P1.5, 1.5-day-old mouse kidney; E17.5 and E15.5, fetal kidney from 17.5 and 15.5-day-old mouse embryo, respectively

Figure 2

Biological evaluation of differentially expressed genes. Comparison of gene expression between Wilms tumor and differentiated kidney. WT: Wilms tumor; DK: differentiated kidney; F: fold change and P: P-value axis Y: Log2 relative expression. Negative and positive values correspond to downregulation and upregulation in WT, respectively. (^*) Represents differentially expressed genes with fold change >3 and P<0.05

Figure 3

Representative sections of immunohistochemical staining in Wilms tumor, fetal and differentiated kidneys. Proteins are identified in each block. Antibody specificity was determined by western blot (panels above graphics). Cell line and molecular weight are indicated. HEK293: human embryonic kidney cell line, SK-NEP-1: Wilms tumor cell line. Graphics: Y axis—protein quantification based on protein intensity values. X axis: Wilms tumor (right) and differentiated kidney (left) samples. A, D, G, J, M, P, S and V: representative samples of Wilms tumor. B, E, H, K, N, Q, T and X: representative samples of fetal kidneys. Fetal kidneys from thirteenth week human embryo: HDGF, IGF2, GRK7, WNT5B and TIMP3; fetal kidneys from seventeenth week human embryo: CRABP2 and FZD2, and; fetal kidneys from the twenty-fourth week human embryo: TESK1. C, F, I, L, O, R, U, Y representative samples of differentiated kidney, only cells from glomerulous were analyzed. WT: Wilms tumor; DK: differentiated kidney. (^*) Represents differentially expressed genes with P<0.05. Black bars represent 100.1 or 100.3 μm as indicated in the green box

Figure 3

Representative sections of immunohistochemical staining in Wilms tumor, fetal and differentiated kidneys. Proteins are identified in each block. Antibody specificity was determined by western blot (panels above graphics). Cell line and molecular weight are indicated. HEK293: human embryonic kidney cell line, SK-NEP-1: Wilms tumor cell line. Graphics: Y axis—protein quantification based on protein intensity values. X axis: Wilms tumor (right) and differentiated kidney (left) samples. A, D, G, J, M, P, S and V: representative samples of Wilms tumor. B, E, H, K, N, Q, T and X: representative samples of fetal kidneys. Fetal kidneys from thirteenth week human embryo: HDGF, IGF2, GRK7, WNT5B and TIMP3; fetal kidneys from seventeenth week human embryo: CRABP2 and FZD2, and; fetal kidneys from the twenty-fourth week human embryo: TESK1. C, F, I, L, O, R, U, Y representative samples of differentiated kidney, only cells from glomerulous were analyzed. WT: Wilms tumor; DK: differentiated kidney. (^*) Represents differentially expressed genes with P<0.05. Black bars represent 100.1 or 100.3 μm as indicated in the green box